Method and contrast agent for use in magnetic resonance imaging of abscess

ABSTRACT

A method and contrast agent for magnetic resonance imaging (MRI) of abscesses. Granulocytes are separated from whole blood and incubated with an imaging vehicle such as polymer-coated magnetite. The granulocytes encapsulate the imaging vehicle and are suspended in an intravascularly administrable carrier to form the contrast agent. The contrast agent is highly specific for MRI of abscesses.

This is a division of copending application Ser. No. 07/238,870 filed onAug. 31, 1988, now U.S. Pat. No. 5,045,304.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The field of this invention is that of contrast agents for magneticresonance imaging (MRI). More specifically, this invention relates to amethod and reagent for use in magnetic resonance imaging of abscesses.

II. Description of the Related Art

Magnetic resonance imaging (MRI) is known as an imaging modality fordiagnosing various conditions in a patient. In order to specificallyimage a particular tissue in the body, it is necessary to direct acontrast agent having paramagnetic or super-paramagnetic properties tothe tissue to be imaged. It is known as disclosed in U.S. Pat. No.4,729,892 to use cross-linked hydrogel materials as contrast agents forimaging gastrointestinal tract. It is also known as disclosed in U.S.Pat. No. 4,669,481 to Eisenberg et al to label red blood cells withchromium for imaging the liver or spleen. It is also known as disclosedin U.S. Pat. No. 4,669,481 to Eisenberg et al. to use metal chelates,such as Gd-DTPA and nitroxide stable radicals (NSFRs) as contrast agentshowever these agents are of limited use because of target specificity,toxicity and relatively short duration signal.

Heretofore, the ability to determine the nature of abscess in a patienthas been accomplished only by computerized tomography and ultra sound.These modalities cannot distinguish an abscess from a neoplastic mass.Because of the non-invasive characteristics of magnetic resonanceimaging, it would be useful to image abscesses by magnetic resonanceimaging. Therefore, it would be advantageous to provide a contrast agentwhich would target abscesses and provide sufficient contrast todistinguish abcess from neoplastic mass.

SUMMARY OF THE PRESENT INVENTION

Accordingly, the present invention consists of a contrast agent for usein magnetic resonance imaging of abscess and a method of preparation anduse of the contrast agent. The contrast agent consists of an imagingvehicle which has been coupled to viable granulocytes. The contrastagent is administered to the patient by intraveneous infusion forimaging of abscess.

The imaging vehicle is an agent which is either paramagnetic,ferro-magnetic, or super-paramagnetic. The imaging vehicle may be formedfrom known paramagnetic metals such as iron, gadolinium, indium, copper,manganese, etc. The paramagnetic metals may be in the form of chelates,such as DTPA; salts; or in the form of elemental metal. The preferredimaging vehicle is magnetite, Fe₂ O₃.FeO which has been coated with apolymer having a high percentage of carboxyl (COOH) groups.

Granulocytes are isolated from the blood of a patient and mixed with anaqueous solution of the imaging vehicle. In the preferred embodiment,the mixture is incubated for a predetermined period of time to permitthe granulocytes to encapsulate the polymer-coated magnetite. Onceencapsulation has taken place, the granulocytes are labelled. Labelledgranulocytes are then suspended in an intravascularly administerableliquid carrier. The labelled granulocytes may be separated from theaqueous solution. The granulocytes are examined microscopically todetermine viability. The suspension of labelled granulocytes isreintroduced into the patient and a specified amount of time is allowedto pass in order for the labelled granulocytes to migrate to abscesssites. The MRI examination is then carried out in the usual manner toobtain an image of the abscess(es). The labelled granulocytes can beobserved using T₁, T₂ and mixed T₁ and T₂ weighted pulse sequences.

EXPERIMENTAL

The method of this invention was tested on an experimental basis usingpolymer-coated magnetite. Magnetite has a formulation of Fe₂ O₃.FeO. Thepolymer-coated magnetite had a high percentage of carboxyl groups(COOH)and a molecular weight of 215.5.

A. Preparation

A commercially available carboxyl terminal polymer-coated magnetite soldby Advanced Magnetics, Inc. of Cambridge, Mass., under the trade name"Biomag" was utilized. The "Biomag" was packaged in a suspension ofwater, having a concentration of 268μ moles Fe/cc. The particles werepelleted from the suspension by centrifugation. The polymer-coatedmagnetite was re-suspended to its original volume in Hank's physiologicbuffer, available from Sigma Corporation, and prepared according topackage directions (pH=7.2) in order to form a suspension ofpolymer-coated magnetite in Hank's physiologic buffer.

Calculations for the amount of agent needed to relax T₁ and T₂ are asfollows:

10 μ/moles of Fe⁺⁺⁺ per kilogram of tissue to be imaged are required toreduce the signal intensity of the T₂ weighted image by 50%. It wasfound that the granulocytes from 100 cc's of blood encapsulated 18.5μmoles of polymer-coated magnetite. Since 18.5μ moles of Biomag contains10⁵ particles Fe⁺⁺⁺ and 100 cc's of blood 0.5×10⁶ granulocytes, eachgranulocyte encapsulates an average of 5 particles. By observation itwas determined that a granulocyte may encapsulate as many as 8 particlesof Fe⁺⁺⁺ . Additionally, since 18.5μ moles of Fe⁺⁺⁺ were encapsulated bythe granulocytes from 100 cc's of blood, 1.85 kilograms of tissue couldbe imaged by the labelled granulocytes.

Whole blood was drawn in heparinized syringes from Sprague-Dawley ratsin order to obtain 50 ccs total volume. The whole blood was thencentrifuged at 400 g's for 30 minutes onto a 50% v/v Ficoll-Hypaquegradient in order to separate granulocytes from the whole blood.

The granulocytes were removed from the gradient and placed in Hank'sphysiologic buffer at a concentration of 200,000 granulocytes per ml.

A solution of 3% Hydroxyethyl Starch (HES) was prepared to facilitateaggregation of the granulocytes. The HES was added to the granulocytesin Hank's physiologic buffer at a concentration of 1 ml HES to 10 mlgranulocytes in Hank's physiologic buffer, to form solution ofgranulocytes and HES in Hank's physiologic buffer.

B. Rat Imaging

Four Sprague-Dawley rats weighing between 428 and 482 gm were injectedwith 0.2 ml of sterile turpentine into the right hind leg, according tothe procedure described in "Preparation and Evaluation of ¹¹¹ In-labeledLeukocytes as an Abscess Imaging Agent in Dogs", Thakur et al, Radiology119: 731-732, June, 1976, in order to artificially create a sterileabcess site. After 24 hours, baseline MR images of the sites wereobtained for each of the four rats.

The solution of prepared granulocytes in Hank's physiologic buffer wasmixed with polymer-coated magnetite (BIOMAG) in Hank's physiolgic bufferat a ratio of 1 ml granulocytes in Hank's physiologic buffer to 0.1 mlpolymer-coated magnetite in Hank's physiologic buffer, to form a coupledsolution.

As known in the art, the coupled solution was incubated with O₂ bubblingthrough at 37 degrees C. for thirty to sixty minutes. The specifiedlength of time was determined by observation of the speed with which thegranulocytes encapsulate or phagatacize one to four particles ofpolymer-coated magnetite. Encapsulation was visually assessed using amicroscope and by staining an aliquot of the granulocytes with an ironstain and/or Wright's stain using standard procedures.

Encapsulation was completed after one to eight polymer-coated magnetiteparticles had been encapsulated by the majority of viable granulocytes.The average encapsulation time is 30 to 60 minutes. The granulocytescontaining the polymer-coated magnetite were considered labelled, andcould be separated from the coupled solution if required using a polemagnet smaller in volume than the mixing vessel in order to isolate thelabelled granulocytes, or contrast agent.

The contrast agent was then injected into each rat at a concentration of200,000 labelled granulocytes per rat. The rats were imaged after 30minutes to 24 hours, in order for the granulocytes to migrate to theabcess sites.

C. Results

Rats were imaged using T₁, T₂ and mixed T₁ and T₂ weighted pulsesequences. Images were compared to baseline.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

According to the invention, the contrast agent for magnetic resonanceimaging of abscess is formed by coupling an imaging vehicle togranulocytes isolated from whole blood. The imaging vehicle may beeither para- and ferro-magnetic in the form of Fe₂ O₃.XO, where X is oneof the known paramagnetic metals, such as iron, gadolinium, indium,copper, or manganese. The paramagnetic metals may be in the form ofchelates, such as DTPA, salts, or elemental metal.

The preferred embodiment for the contrast agent is a suspension ofgranulocytes which have encapsulated or phagocytised polymer-coatedmagnetite. According to the method of the invention, the suspension isformed in the manner disclosed in the experimental section above.

In the preferred embodiment, the imaging vehicle is polymer-coatedmagnetite, magnetite being Fe₂ O₃.FeO. The magnetite is coated withpolymer to facilitate encapsulation by the granulocytes. The magnetiteis coated with a polymer of carbon-link chains having functionalterminal groups consisting of carboxyl (COOH), amine (NH₂) or otherfunctional chemicals. A suitable polymer-coated magnetite is availablefrom Advance Magnetics, Inc. of Cambridge, Mass., and sold under thetrade name "Biomag". BIOMAG is available in a suspension of water havinga concentration of 268μ moles Fe/cc and a molecular weight of 215.5. Thewater was removed from the suspension by centrifugation, as is known inthe art. The particles of polymer-coated magnetite were re-suspended in0.1 to 0.5 ml of Hank's physiologic buffer. Hank's physiologic buffer isavailable from Sigma Corporation, and the suspension was preparedaccording to package directions (pH=7.2) to form a suspension ofpolymer-coated magnetite (BIOMAG particles) in Hank's physiologicbuffer.

A supply of the granulocytes is then isolated from whole blood. In thepreferred embodiment, whole blood is drawn from the person or animalwhose tissue is to be imaged. Whole blood is drawn in heparinizedsyringes to obtain a suitable supply of granulocytes. 500,000granulocytes may be isolated from 100 cc's of blood. The 500,000granulocytes will encapsulate 1.85μ moles of Biomag to produce asufficient quantity of contrast agent to image 1.85. kilograms oftissue.

A supply of heparinized blood is then mixed with a 3% HydroxyethylStarch (HES) solution, available from McGraw Laboratories, Chicago, Ill.The solution is formed in a ratio of 100 cc's of blood to 20 cc's of the3% HES. The solution is permitted to stand for 30 to 60 minutes to anhour and a half. The HES solution facilitates aggregation of thegranulocytes as the red blood cells will rouleaux, as known in the art.The serum containing the granulocytes is then drawn off from therouleauxed blood and centrifuged at 250 g's for five minutes. Thegranulocytes are then removed by suitable means such as pipetting andre-suspended in a 1 to 2 mls volume Hank's physiologic buffer.

The Hank's solution containing the imaging vehicle (I), is combined witha solution of granulocytes in Hank's (II) at a ratio of 0.1 to 0.5 ml(I) to 1 to 2 ml (II), thus forming a coupled solution. The coupledsolution is then incubated at 37° C. The coupled solution is oxygenatedby blowing O₂ on the surface of the solution during incubation in orderto preserve the viability of the granulocytes. The granulocytesencapsulate or phagocytise a polymer-coated magnetite in 30 to 60minutes. After 30 minutes, approximately 90% of the granulocytes willhave encapsulated at least 3 particles of polymer-coated magnetite. By60 minutes, each of the granulocytes has encapsulated or phagacytized 3to 8 particles. The labelled granulocytes may be separated from theaqueous solution of physiologic buffer in unlabelled granulocytes. Onemethod of separation is inserting a pole magnet smaller than the vesselcontaining the coupled solution to attract the labelled granulocytes.However, since 90% of the granulocytes successfully encapsulate thepolymer-coated magnetite particles, it is often not necessary toseparate the labelled granulocytes from the non-labelled granulocytes.Encapsulation may be visually accessed by using a microscope and bystaining an aliquot of the granulocytes with an iron stain and/orWright's stain using known procedures.

Viable labelled granulocytes are then separated from the solution,washed of debris and re-suspended in an intervasculatory admissablecarrier such as plasma or physiological saline to form a contrast agent.The contrast agent must be administered within a few hours ofpreparation in order to insure the viability of the granulocytes. Thecontrast agent is suspended in a suitable equipped carrier and isadministered to the patient or animal to be imaged by intravenousinfusion. The same techniques and procedures may be followed as are usedin the regular administration of blood. Time is permitted to elapse inorder to permit the granulocytes to travel to the tissue to be imaged.This length of time is normally in the range of approximately 30 minutesto 24 hours. After the waiting period has elapsed, the patient issubjected to a standard MRI examination.

The MRI examination is carried out in a known manner with respect to theparticular purpose of the examination. In general, the post sequenceswill be selected to maximize either contrast induced changes in T₁ orT₂. T₁ and T₂ current tissue primers to determine saline intensity ofMRI imaging. The appearance of a sharply delineated dark area in thetissue is interpreted to be a highly specific image of the abscess.Thus, abscess can be distinguished from neoplastic mass by observing adecrease in signal intensity in T₂ weighted images.

It is contemplated that other reagents may be formed in accordance withthe invention. Isolated granulocytes may be coupled with other imagingvehicles such as paramagnetic metals such as iron, gadolinium, indium,copper, manganese, etc. These paramagnetic metals may be coupled to thegranulocytes by phagocytation, studding or other suitable manners ofcoupling.

It should be apparent to one having ordinary skill in the art that manymodifications or changes can be made to the preferred embodiment withoutdeparting from the spirit of the present invention as expressed in thescope of the appended claims.

We claim:
 1. A method of preparing and using a contrast agent inmagnetic resonance imaging of abscesses in a patient comprising thesteps of:mixing a specific number of viable granulocytes with an aqueoussolution of a polymer-coated metallic imaging agent in which saidimaging agent binds to and is subsequently incorporated into thegranulocytes incubating said aqueous solution to encapsulate saidimaging agent in said granulocytes to form labeled granulocytes;separating said labeled granulocytes from said solution; suspending saidlabeled granulocytes in an intravascularly administerable carrier toform said contrast agent; administering a diagnostically effectiveamount of said contrast agent by intravenous infusion to said patient;and subjecting said patient to examination by magnetic resonanceimaging.
 2. The method of claim 1 in which said imaging vehicle ispolymer-coated magnetite; Fe₂ O₃.FeO.
 3. A contrast reagent for MRI ofabscesses, said reagent comprising;a suspension of labeled granulocytesin an intravascularly administrable carrier, said granulocytes bindingto and subsequently incorporating within a diagnostically effectiveamount of an imaging agent, said agent having a polymer coating, andhaving the formula Fe₂ O₃.XO where X is one of the group consisting ofpara-magnetic and ferro-magnetic materials.
 4. The contrast agent ofclaim 3 wherein said imaging vehicle is polymer-coated magnetite, (Fe₂O₃.FeO).
 5. The contrast agent of claim 4 wherein said polymer-coatedmagnetite was encapsulated with said granulocytes.